D Zoukhri1, R R Hodges, C Sergheraert, D A Dartt. 1. Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA. zoukhri@vision.eri.harvard.edu
Abstract
PURPOSE: To investigate the role of protein kinase C (PKC) in cholinergic agonist-induced Ca2+ elevation in lacrimal gland acini. METHODS: Lacrimal gland acini were prepared by collagenase digestion, and changes in intracellular Ca2+ ([Ca2+]i) were measured using fura-2 as a fluorescent probe. RESULTS: Preactivation of PKC by phorbol 12-myristate 13-acetate (PMA), or inhibition of protein phosphatase type 1/2A (PP1/2A) by calyculin A, decreased both the [Ca2+]i transient and the plateau of [Ca2+]i induced by increasing concentrations of carbachol, a cholinergic agonist. Staurosporine, an inhibitor of PKC, completely reversed the effect of PMA. Inhibition of the Ca(2+)-independent PKC isoforms PKCdelta and -epsilon, but not the Ca(2+)-dependent isoform PKCalpha substantially reversed the inhibitory effect of PMA on cholinergic agonist-induced Ca2+ elevation. The inhibitory effect of PMA was obtained only in the presence of extracellular Ca2+, suggesting that PKC inhibits the influx of Ca2+. PMA completely inhibited the cholinergic agonist-induced plateau of [Ca2+]i. PMA and calyculin A decreased both the [Ca2+]i transient and the plateau of [Ca2+]i induced by thapsigargin, further supporting the idea that PKC modulates the entry of Ca2+. CONCLUSIONS: In the lacrimal gland, agonist-induced changes in [Ca2+]i are negatively regulated by PKC-dependent phosphorylation of a target protein(s) that is sensitive to PP1/2A.
PURPOSE: To investigate the role of protein kinase C (PKC) in cholinergic agonist-induced Ca2+ elevation in lacrimal gland acini. METHODS: Lacrimal gland acini were prepared by collagenase digestion, and changes in intracellular Ca2+ ([Ca2+]i) were measured using fura-2 as a fluorescent probe. RESULTS: Preactivation of PKC by phorbol 12-myristate 13-acetate (PMA), or inhibition of protein phosphatase type 1/2A (PP1/2A) by calyculin A, decreased both the [Ca2+]i transient and the plateau of [Ca2+]i induced by increasing concentrations of carbachol, a cholinergic agonist. Staurosporine, an inhibitor of PKC, completely reversed the effect of PMA. Inhibition of the Ca(2+)-independent PKC isoforms PKCdelta and -epsilon, but not the Ca(2+)-dependent isoform PKCalpha substantially reversed the inhibitory effect of PMA on cholinergic agonist-induced Ca2+ elevation. The inhibitory effect of PMA was obtained only in the presence of extracellular Ca2+, suggesting that PKC inhibits the influx of Ca2+. PMA completely inhibited the cholinergic agonist-induced plateau of [Ca2+]i. PMA and calyculin A decreased both the [Ca2+]i transient and the plateau of [Ca2+]i induced by thapsigargin, further supporting the idea that PKC modulates the entry of Ca2+. CONCLUSIONS: In the lacrimal gland, agonist-induced changes in [Ca2+]i are negatively regulated by PKC-dependent phosphorylation of a target protein(s) that is sensitive to PP1/2A.
Authors: Shivaram Selvam; Padmaja B Thomas; Sarah F Hamm-Alvarez; Joel E Schechter; Douglas Stevenson; Austin K Mircheff; Melvin D Trousdale Journal: Adv Drug Deliv Rev Date: 2006-09-15 Impact factor: 15.470
Authors: Nora Botten; Robin R Hodges; Dayu Li; Jeffrey A Bair; Marie A Shatos; Tor P Utheim; Charles N Serhan; Darlene A Dartt Journal: FASEB J Date: 2019-04-23 Impact factor: 5.191